Chlamydial Growth Research Articles

Foam cell formation inhibits growth of Chlamydia pneumoniae but does not attenuate Chlamydia pneumoniae-induced secretion of proinflammatory cytokines.

It has not yet been determined whether lipid-loaded macrophages (foam cells), a major cellular component of atherosclerotic lesions, have the capacity to support growth of Chlamydia pneumoniae and be activated to secrete proinflammatory cytokines in response to C pneumoniae infection. Lipid loading of RAW 264.7 cells and mouse peritoneal macrophages with either oxidized or acetylated LDL significantly inhibits the growth of C pneumoniae. Modified forms of LDL are not directly toxic to C pneumoniae and do not inhibit either the initial binding or internalization of C pneumoniae by macrophages. Lipid loading does not reduce infection of macrophages with Chlamydia trachomatis. Treatment of lipid-loaded macrophages with live, heat-killed, or UV-inactivated C pneumoniae stimulates secretion of cytokines. C pneumoniae also induces expression of the mRNA for tumor necrosis factor-alpha in foam cells despite inhibition of nuclear factor-kappaB binding to DNA by prior treatment with oxidized LDL. Foam cell formation is not conducive to growth of C pneumoniae but does not inhibit the C pneumoniae-induced secretion of proinflammatory cytokines.

The growth cycle of Simkania negevensis.

Simkania negevensis, a bacterium formerly referred to as 'the micro-organism Z' or 'Simkania Z', belongs to the order Chlamydiales, assigned to the family Simkaniaceae: The purpose of this study was to investigate the production of Simkania negevensis progeny in infected cells in comparison with the well-documented Chlamydiaceae developmental cycle. It was found that replicating Simkania negevensis in Vero cells resembled the reticulate bodies of all known chlamydial species: in electron micrographs they were reticulated, homogeneously staining, and often caught in the process of binary division. These replicative forms were found in low abundance shortly after infection, but by 3 days post-infection they were the most prevalent particles in host cells. Electron-dense forms of Simkania negevensis began to appear on the third day post-infection, but quantitatively did not account for the high titre of infectivity in extracts from these host cells. These had both electron-dense and electron-lucent areas, a characteristic seen only in a few chlamydial species. Simkania negevensis infectivity did not appreciably change during the ensuing 12 days required for host cell lysis, despite an eightfold increase in the proportion of electron-dense bacteria over this time. The emergence of electron-dense bodies, increase in infectivity and host-cell lysis were not synchronized developmental events. This is a novel finding in Chlamydiales spp. and suggests that Simkania negevensis will provide new perspectives in the mechanisms of chlamydial intracellular growth.

Novel acoustic azithromycin-encapsulated liposomes inhibit Chlamydial growth in endothelial cells

Novel acoustic azithromycin-encapsulated liposomes inhibit Chlamydial growth in endothelial cells

Fc receptor regulation of protective immunity against Chlamydia trachomatis

The prevailing paradigm for designing potentially efficacious vaccines against the obligate intracellular bacterium, Chlamydia trachomatis, advocates regimens capable of inducing a mucosal antigen-specific T helper type 1 (Th1) response. However, recent reports indicate that rapid and efficient clearance of a secondary infection also requires certain B-cell functions. We investigated the hypothesis that Fc receptor (FcR)-mediated antibody effector mechanisms are important B-cell-related functions involved in controlling a chlamydial genital reinfection. Microbiological analysis of genital chlamydial infection in FcR knockout (FcRKO) mice lacking the activatory FcgammaRI (CD64) and FcRgammaIII (CD16), as well as the inhibitory FcgammaRIIB1 (CD32), revealed a greater intensity of secondary infection (i.e. bacterial shedding) in FcRminus sign/minus sign as compared to FcR+/+ mice; however, the course of the primary infection was indistinguishable in both animals. Pathologically, FcRKO mice suffered greater ascending infection than immunocompetent wild-type (WT) mice after a secondary infection. Immunological evaluation indicated that the presence of specific anti-chlamydial antibodies enhanced chlamydial antigen presentation for induction of a Th1 response by FcR+/+, but not FcRminus sign/minus sign, antigen-presenting cells. In addition, specific anti-chlamydial antibodies augmented both macrophage killing of infected epithelial cells by antibody-dependent cellular cytotoxicity (ADCC) and macrophage inhibition of productive growth of chlamydiae in co-cultures. These results indicate that B cells participate in anti-chlamydial immunity via FcR-mediated effector functions of antibodies, which are operative during reinfections. Such effector functions include ADCC, and possibly enhanced uptake, processing and presentation of chlamydial antigens for rapid induction of a Th1 response, all facilitating the early clearance of an infection. These findings suggest that a future anti-chlamydial vaccine should elicit both humoral and T-cell-mediated immune responses for optimal memory response and vaccine efficacy.

Interferon-β induction by Chlamydia pneumoniae in human smooth muscle cells

Clinical studies have suggested a causal or contributory role of Chlamydia pneumoniae infection in asthma and atherosclerosis. The activation of synthetic functions of smooth muscle cells (SMC) including the production of cytokines and growth factors plays a major role in the formation of fibrous atherosclerotic plaques as well as in structural remodelling of the airway wall in chronic asthma. In this study we demonstrated that C. pneumoniae induced the production of low levels of interferon (IFN)-β in bronchial and vascular SMC when infected cells were treated with tumour necrosis factor-α (TNF-α). IFN-β production was analysed by reverse transcription-PCR and enzyme-linked immunosorbent assay. The upregulation of IFN-β was paralleled by an increase in mRNA levels of interferon regulatory factor-1 and interferon-stimulated gene factor 3γ, two transcription factors activating the expression of the IFN-β gene. In addition, C. pneumoniae infection enhanced the mRNA level of indoleamine 2,3-dioxygenase, an IFN-inducible factor mediating the restriction of intracellular chlamydial growth, in TNF-α-stimulated SMC. C. pneumoniae-induced IFN-β production by SMC may modulate inflammation and tissue remodelling during respiratory and vascular infection.

Interferon-beta induction by Chlamydia pneumoniae in human smooth muscle cells.

Clinical studies have suggested a causal or contributory role of Chlamydia pneumoniae infection in asthma and atherosclerosis. The activation of synthetic functions of smooth muscle cells (SMC) including the production of cytokines and growth factors plays a major role in the formation of fibrous atherosclerotic plaques as well as in structural remodelling of the airway wall in chronic asthma. In this study we demonstrated that C. pneumoniae induced the production of low levels of interferon (IFN)-beta in bronchial and vascular SMC when infected cells were treated with tumour necrosis factor-alpha (TNF-alpha). IFN-beta production was analysed by reverse transcription-PCR and enzyme-linked immunosorbent assay. The upregulation of IFN-beta was paralleled by an increase in mRNA levels of interferon regulatory factor-1 and interferon-stimulated gene factor 3gamma, two transcription factors activating the expression of the IFN-beta gene. In addition, C. pneumoniae infection enhanced the mRNA level of indoleamine 2,3-dioxygenase, an IFN-inducible factor mediating the restriction of intracellular chlamydial growth, in TNF-alpha-stimulated SMC. C. pneumoniae-induced IFN-beta production by SMC may modulate inflammation and tissue remodelling during respiratory and vascular infection.

Chlamydia trachomatis persistence in the female mouse genital tract: inducible nitric oxide synthase and infection outcome.

It was previously reported that female mice resolve a primary Chlamydia trachomatis urogenital infection independent of inducible nitric oxide synthase (iNOS). We now report that although iNOS-deficient (NOS2(-/-)) mice resolve culture-apparent infection in a fashion similar to that of normal control (NOS2(+/+)) mice, they sustain significantly increased rates of disease, as assessed by hydrosalpinx formation. PCR amplification of ompA followed by Southern blot detection of amplicands revealed the presence of chlamydial DNA in the lower genital tracts of both NOS2(-/-) and NOS2(+/+) mice at > or =120 days postinfection and in upper genital tract tissues at >120 days postinfection. However, only NOS2(-/-) mice shed low numbers of viable chlamydiae from the lower genital tract after immunosuppressive treatment at 120 days postinfection. When cultured primary murine lung fibroblasts were activated in the presence of gamma interferon (IFN-gamma), inhibition of chlamydial growth occurred in both NOS2(+/+) and NOS2(-/-) cells, but the inhibition was reversible after removal of the cytokine in the NOS2(-/-) primary cell culture only. The iNOS-independent inhibition was microbistatic but was independent of 2,3-indoleamine dioxygenase activity. We conclude that chlamydial DNA and antigens persist in mice subsequent to culture-apparent resolution. In addition, IFN-gamma induces in vivo inhibition of chlamydial growth through microbistatic mechanisms in the absence of iNOS activity, but in the presence of iNOS activity, IFN-gamma is microbicidal and effects eradication.

Inhibition in vitro de la croissance de Chlamydia trachomatis par des cyclines encapsulées dans les liposomes

The antichlamydial activity of tetracycline (Tet) and doxycycline (Dox) encapsulated in cationic (CaL), anionic (AnL) and neutral (NtL) liposomes has been evaluated in vitro by adding serial dilutions of antibiotics (minimum inhibitory concentration, MIC: 0.12–0.007 μg/ml; MBC: 4 to 0.25 μg/ml) to HeLa 229 cell monolayers inoculated with Chlamydia trachomatis L 2/434/Bu (10 3 ufi/ml). Following 72 h incubation at 37°C under a 5% CO 2 atmosphere, the chlamydial inclusions were stained by the May-Giemsa method to determine the MICs. After a second and third passage, the MBC 1 and MBC 2 were determined in antibiotic-free medium. The chlamydial inclusions were then counted to assess the degree of growth inhibition at each antibiotic dilution tested for MBC 1 and MBC 2 determinations. The MIC, MBC 1 and MBC 2 of the various antichlamydial agents were as follows: Tet (0.12; 4; 4), AnL-Tet (0.01; 1; 1), NtL-Tet (0.03; 1; 2), Dox (0.06; 1; 2), CaL-Dox (0.03; 0.5; 2), AnL-Dox (0.01; 1; 2), and NtL-Dox (0.03; 0.5; 0.5). It was found that Tet and Dox liposome-encapsulated antibiotics were more active than their non-encapsulated counterparts, and the inclusion count showed a higher inhibitory activity of the former antibiotics on chlamydial growth. The inhibition of chlamydial growth by AnL-Tet may be of bactericidal nature. In conclusion, liposome-encapsulated drugs could be of value in the treatment of chlamydial infections

Chlamydia pneumoniae infection in circulating human monocytes is refractory to antibiotic treatment.

Recovery of the intracellular bacterium Chlamydia pneumoniae from atherosclerotic plaques has initiated large studies on antimicrobial therapy in coronary artery disease. The basic concept that antibiotic therapy may eliminate and prevent vascular infection was evaluated in vitro and in vivo by examining the antibiotic susceptibility of C pneumoniae in circulating human monocytes, which are thought to transport chlamydiae from the respiratory tract to the vascular wall. Blood monocytes (CD14+) from 2 healthy volunteers were obtained before and after oral treatment with azithromycin or rifampin and then inoculated with a vascular C pneumoniae strain and continuously cultured in the presence of the respective antibiotic. Progress of infection and chlamydial viability was assessed by immunogold-labeling and detection of C pneumoniae-specific mRNA transcripts. Circulating monocytes from patients undergoing treatment with experimental azithromycin for coronary artery disease were examined for C pneumoniae infection by cell culture. Antibiotics did not inhibit chlamydial growth within monocytes. Electron microscopy showed development of chlamydial inclusion bodies. Reverse transcription-polymerase chain reaction demonstrated continuous synthesis of chlamydial mRNA for 10 days without lysis of the monocytes. The in vivo presence of viable pathogen not eliminated by azithromycin was shown by cultural recovery of C pneumoniae from the circulating monocytes of 2 patients with coronary artery disease. C pneumoniae uses monocytes as a transport system for systemic dissemination and enters a persistent state not covered by an otherwise effective antichlamydial treatment. Prevention of vascular infection by antichlamydial treatment may be problematic: circulating monocytes carrying a pathogen with reduced antimicrobial susceptibility might initiate reinfection or promote atherosclerosis by the release of proinflammatory mediators.

Modulation of P2Z/P2X(7) receptor activity in macrophages infected with Chlamydia psittaci.

Given the role that extracellular ATP (ATP(o))-mediated apoptosis may play in inflammatory responses and in controlling mycobacterial growth in macrophages, we investigated whether ATP(o) has any effect on the viability of chlamydiae in macrophages and, conversely, whether the infection has any effect on susceptibility to ATP(o)-induced killing via P2Z/P2X(7) purinergic receptors. Apoptosis of J774 macrophages could be selectively triggered by ATP(o), because other purine/pyrimidine nucleotides were ineffective, and it was inhibited by oxidized ATP, which irreversibly inhibits P2Z/P2X(7) purinergic receptors. Incubation with ATP(o) but not other extracellular nucleotides inhibits the growth of intracellular chlamydiae, consistent with previous observations on ATP(o) effects on growth of intracellular mycobacteria. However, chlamydial infection for 1 day also inhibits ATP(o)-mediated apoptosis, which may be a mechanism to partially protect infected cells against the immune response. Infection by Chlamydia appears to protect cells by decreasing the ability of ATP(o) to permeabilize macrophages to small molecules and by abrogating a sustained Ca(2+) influx previously associated with ATP(o)-induced apoptosis.

Host cell-derived sphingolipids are required for the intracellular growth of Chlamydia trachomatis.

Chlamydia trachomatis, an important cause of human disease, is an obligate intracellular bacterial pathogen that relies on the eukaryotic host cell for its replication. Recent reports have revealed that the C. trachomatis vacuole receives host-derived sphingolipids by fusing with trans-Golgi network (TGN)derived secretory vesicles. Here, it is shown that these lipids are required for the growth of the bacteria. C. trachomatis was unable to replicate at 39 degrees C in the Chinese hamster ovary (CHO)-derived cell line SPB-1, a cell line incapable of synthesizing sphingolipids at this temperature because of a temperature-sensitive mutation in the serine palmitoyltransferase (SPT) gene. Complementation with the wild-type SPT gene or addition of exogenous cell-permeable sphingolipid precursors to the mutant cells restored their ability to support chlamydial replication. L-cycloserine (L-CS) and fumonisin B1 (FB1), inhibitors of sphingolipid biosynthesis, decreased the proliferation of the bacteria in eukaryotic cells at concentrations that also decreased host cell sphingolipid synthesis. In the case of FB1, the vacuoles appeared aberrant; the addition of sphingolipid precursors was able to reverse the altered morphology of the FB1-treated vacuoles. Collectively, these data strongly suggest that the growth and replication of chlamydiae is dependent on synthesis of sphingolipids by the eukaryotic host cell and may contribute to this organism's obligate intracellular parasitism.

Persistence of Chlamydia trachomatis is induced by ciprofloxacin and ofloxacin in vitro.

An in vitro cell culture model was used to investigate the long-term effect of ciprofloxacin and ofloxacin on infection with Chlamydia trachomatis. Standard in vitro susceptibility testing clearly indicated successful suppression of chlamydial growth. To mimic better in vivo infection conditions, extended treatment with the drugs was started after infection in vitro had been well established. Incubation of such established chlamydial cultures with ciprofloxacin and ofloxacin not only failed to eradicate the organism from host cells, but rather induced a state of chlamydial persistence. This state was characterized by the presence of nonculturable, but fully viable, bacteria and the development of aberrant inclusions. In addition chlamydia exhibited altered steady-state levels of key chlamydial antigens, with significantly reduced major outer membrane protein and near constant hsp60 levels. Resumption of overt chlamydial growth occurred after withdrawal of ciprofloxacin, confirming the viability of persisting chlamydia. In vitro ciprofloxacin results are consistent with clinical data, thereby providing an explanation for treatment failures of ciprofloxacin. Parallel in vitro studies with ofloxacin suggest a better correlation between clinical and laboratory-defined efficacy, although the clinical studies on which this assessment is based did not include monitoring of chlamydial persistence. The data presented here clearly demonstrate that under at least some circumstances, standard determination of MICs and minimal bactericidal concentrations for C. trachomatis allows no more than a simple definition of whether an antibiotic has some anti chlamydial activity; however, such testing is not always sufficient to verify that the antibiotic will eliminate the organism in vivo.

Differential sensitivities of Chlamydia trachomatis strains to inhibitory effects of gamma interferon.

Gamma interferon (IFN-gamma) is an important cytokine in host defense against chlamydial infection. An in vitro cell culture system was used to show that IFN-gamma inhibition of chlamydial growth, as determined by diminished recovery of infectious elementary bodies, differed markedly among chlamydial strains. These differences in sensitivity among chlamydial strains to IFN-gamma-mediated inhibition may profoundly influence the clinical outcome of infection.

Frequent contamination of Chlamydia trachomatis and Chlamydia pneumoniae strains with mycoplasma. Biological relevance and selective eradication of mycoplasma from chlamydial cultures with mupirocin.

Several strains of Chlamydia trachomatis (CT) and C. pneumoniae (CP) from different sources were screened for mycoplasma contamination using a sensitive nested 16S rDNA polymerase chain reaction-specific for a broad range of mycoplasma species. Five of nine CT and 5/16 CP isolates were contaminated by mycoplasma. Mycoplasma fermentans, M. hyorhinis and M. hominis were found as contaminating agents. To our knowledge no data are available on whether coinfection of chlamydia with mycoplasma alters the biological behavior of chlamydia. Analysis of the biological effect of mycoplasma on chlamydial infection showed a profound mycoplasma-induced reduction of chlamydial growth. Mycoplasma were efficiently eliminated from chlamydial cultures in HEp-2 cells by treatment with mupirocin without affecting chlamydial replication or host cell growth. Two chlamydial strains, C. trachomatis serovar K and one clinical isolate of C. pneumoniae were purged by this method.

Dynamics of the development of Chlamydophila psittaci inclusions in epithelial and fibroblast host cells.

The development of Chlamydophila psittaci (formerly Chlamydia psittaci, avian strains) inclusions in fibroblast L-929 and epithelial BGM cell lines was studied along the bacterial growth cycle using a BGM cell-adapted strain in the presence or absence of cycloheximide and cycloheximide + polyethylene glycol. Evolution of the inclusions was determined in terms of their number and size at 24, 30, 36, 48 and 54 h after infection. Significant differences in the chlamydial growth were found between both host cells, throughout the study. Higher numbers of inclusions (P < 0.05) were observed in L cells while larger inclusions (P < 0.01) were found in BGM cells. In both fibroblast and epithelial cells, inclusions showed a significant (P < 0.001) increase in size at the later times studied. Free extracellular chlamydial particles were noticed at 48 and 54 h post-infection (p.i.) in infected L cells, and at 54 h p.i. in BGM cells. Addition of cycloheximide or cycloheximide + polyethylene glycol had no significant effect on the number of inclusions or their size. The results suggest that host cell characteristics and innate compatibility between Chlamydophila strain and host cell are more important than host cell adaptation for the development of the microorganism.

Isolation and continuous growth of Chlamydia pneumoniae from arterectomy specimens.

For the purpose of collecting Chlamydia pneumoniae strains of vascular origin that could be grown continuously in vitro, a cell culture system has been established. Using different types of vascular specimens obtained from 38 patients, Chlamydia pneumoniae could be isolated in three (7.9%) cases. The strains were obtained from specimens of the carotid artery, the femoral artery and an infrarenal aneurysm of the abdominal aorta of three male atherosclerosis patients. Thus, viable Chlamydia pneumoniae strains are also present in vascular regions other than those hitherto described.

Tumor necrosis factor-alpha and lipopolysaccharide enhance interferon-induced antichlamydial indoleamine dioxygenase activity independently.

In macrophages, interleukin-1 (IL-1) and lipopolysaccharide (LPS) enhance the antichlamydial effect of interferon-gamma (IFN-gamma) by increasing indoleamine 2,3-dioxygenase (IDO) activity in a dose-dependent manner. Our objectives were to characterize the antichlamydial effect of tumor necrosis factor-alpha (TNF-alpha) on IFN-induced IDO activity and to establish the relationship between LPS and TNF-alpha in IDO potentiation. TNF-alpha inhibited chlamydial growth in a dose-dependent manner only in IFN-treated macrophages. Furthermore, excess tryptophan reversed the effect of combined cytokine treatment, indicating that IDO alone was responsible for chlamydial inhibition. The promonocyte THP-1 cell line, previously used to model the effect of IL-1 on IDO mRNA expression, was treated with IFN-gamma and increasing concentrations of LPS or TNF-alpha. IDO mRNA was quantified by RT-PCR, and IDO activity was measured by HPLC at 24 and 48 h after treatment, respectively. Both LPS and TNF-alpha enhanced IDO activity and IDO mRNA expression, with maximal IDO induction at 100 ng/ml LPS or 5 ng/ml TNF-alpha. Anti-TNF-alpha failed to neutralize the effects of LPS treatment, and insufficient TNF-alpha or IL-1 was produced by LPS-treated THP-1 cells to account for the enhancing effect of LPS, indicating that the effect of LPS on IDO was independent of TNF-alpha and IL-1.

Interaction between turkey monocytes and avian Chlamydia psittaci in the presence of Mycoplasma sp.: the importance of nitric oxide

The interaction between Chlamydia psittaci and turkey monocytes was studied in vitro. Purified monocytes were inoculated with C. psittaci, in the presence or absence of Mycoplasma hyorhinis. Whereas turkey monocytes produced high amounts of nitric oxide (NO) following the inoculation with M. hyorhinis, inoculation with C. psittaci did not induce NO production in these phagocytes. The monocytes strongly supported chlamydial growth, as demonstrated by the presence of inclusion forming units, the positive direct immunofluorescence staining and transmission electron microscopy. In contrast, upon co-inoculation of the monocytes with C. psittaci and M. hyorhinis, a reduced replication rate of C. psittaci was observed. N G-monomethyl- L-Arginine, a competitive inhibitor for the enzyme NO-synthase, inhibited the NO production and reversed the antichlamydial activity of the M. hyorhinis co-inoculated turkey monocytes. These results imply two considerations. First, as chlamydiae are obligate intracellular bacteria, special care should be taken to guard chlamydial cultures from mycoplasmal contamination, in order to prevent false results when investigating the response of immunomodulating cells to chlamydial infection. Secondly, as a mycoplasmal co-infection in vitro has the capacity of inducing antichlamydial activity in turkey monocytes, through the action of NO, it could be suggested that a similar interaction might take place in vivo. Moreover, it was shown that avian M. gallisepticum strains were also able to induce NO in turkey monocytes. Considering the high prevalence of both C. psittaci and Mycoplasma sp. in turkeys, this interaction, through the pivotal role of NO, might influence the outcome of respiratory diseases in turkeys.

The resistance of human monocyte-derived macrophages to Chlamydia pneumoniae infection is enhanced by interferon-gamma.

Chlamydia pneumoniae is an intracellular bacterium which commonly causes respiratory infections. Chronic infections have been associated with atherosclerosis and the organism has been detected in macrophages in the disease lesions. Growth of chlamydiae in different epithelial cell lines is restricted by interferon-gamma (IFN-gamma), a monocyte activator produced by T cells. We have studied the influence of IFN-gamma on the growth and infectivity of C. pneumoniae in HL-cells and human monocyte-derived macrophages. Low concentrations of the cytokine significantly restricted the growth and productivity of C. pneumoniae in epithelial cells in vitro. In macrophages, however, no effect on the growth of the bacteria in infected cells was found, but high doses clearly restricted the production of infectious progeny. The results suggest that IFN-gamma participates in the resistance to C. pneumoniae. The bacterium is, however, still capable of infecting macrophages that are important in the pathogenesis of atherosclerosis and it may thus participate in the inflammatory process associated with the disease.

Characterization and intracellular trafficking pattern of vacuoles containing Chlamydia pneumoniae in human epithelial cells.

Chlamydiae are obligate intracellular pathogens that reside within a membrane-bound vacuole throughout their developmental cycle. In this study, the intraphagosomal pH of Chlamydia pneumoniae (Cpn) was qualitatively assessed, and the intracellular fate of the pathogen-containing vacuole and its interaction with endocytic organelles in human epithelial cells were analysed using conventional immunofluorescence and confocal microscopy. The pH-sensitive probes acridine orange (AO), LysoTracker (LyT) and DAMP did not accumulate in the bacterial inclusion. In addition, exposure of cells to bafilomycin A1(BafA1), a potent acidification inhibitor, did not inhibit or delay chlamydial growth. The chlamydial compartment was not accessible to the fluid-phase tracer Texas Red (TR)-dextran and did not exhibit any level of staining for the late endosomal marker cation-independent mannose-6-phosphate receptor (Ci-M6PR) or for the lysosomal-associated membrane proteins (LAMP-1 and -2) and CD63. In addition, transferrin receptor (TfR)-enriched vesicles were observed close to Cpn vacuoles, potentially indicating a specific translocation of these organelles through the cytoplasm to the vicinity of the vacuole. We conclude that Cpn, like other chlamydial spp., circumvents the host endocytic pathway and inhabits a non-acidic vacuole, which is dissociated from late endosomes and lysosomes, but selectively accumulates early endosomes.